Heparan Sulfate Phage Display Antibodies Identify Distinct Epitopes with Complex Binding Characteristics

Abstract

Heparan sulfate (HS) binds and modulates the transport and activity of a large repertoire of regulatory proteins. The HS phage display antibodies are powerful tools for the analysis of native HS structure in situ; however, their epitopes are not well defined. Analysis of the binding specificities of a set of HS antibodies by competitive binding assays with well defined chemically modified heparins demonstrates that O-sulfates are essential for binding; however, increasing sulfation does not necessarily correlate with increased antibody reactivity. IC 50 values for competition with double modified heparins were not predictable from IC 50 values with corresponding singly modified heparins. Binding assays and immunohistochemistry revealed that individual antibodies recognize distinct epitopes and that these are not single linear sequences but families of structurally similar motifs in which subtle variations in sulfation and conformation modify the affinity of interaction. Modeling of the antibodies demonstrates that they possess highly basic CDR3 and surrounding surfaces, presenting a number of possible orientations for HS binding. Unexpectedly, there are significant differences between the existence of epitopes in tissue sections and observed in vitro in dot blotted tissue extracts, demonstrating that in vitro specificity does not necessarily correlate with specificity in situ/ vivo. The epitopes are therefore more complex than previously considered. Overall, these data have significance for structure-activity relationships of HS, because the model of one antibody recognizing multiple HS structures and the influence of other in situ HS-binding proteins on epitope availability are likely to reflect the selectivity of many HS-protein interactions in vivo.HS 2 belongs to the glycosaminoglycan family of polysaccharides and can be considered the most information-rich biopolymer in nature. HS chains consist of repeating ␤-D-glucuronic acid and GlcNAc units, which are subject to enzymatic postpolymeric modification by N-deacetylation and N-sulfation of GlcNAc, O-sulfation at various positions, and epimerization of D-glucuronic acid to its C-5 epimer, iduronic acid. HS biosynthetic enzymes do not act upon every potential site within a chain, resulting in chains with a high level of structural diversity and a distinct domain structure, with regions of unmodified saccharides (N-acetyl domains) and highly modified saccharides (N-sulfated domains) flanked by regions of alternating modified and unmodified disaccharides (transition or N-acetyl/ N-sulfated domains) (1-4). HS chains are usually attached to core proteins, forming HS proteoglycans, and these proteoglycans represent one of the major components of most metazoan cell surfaces and extracellular matrices. Here, they control cellcell and cell-matrix communication by association with a large repertoire of regulatory proteins (5), including growth factors, morphogens, extracellular matrix components, enzymes, cell adhesion molecules, and cytokines. It is through these int...

Heparan sulfate phage display antibodies recognise epitopes defined by a combination of sugar sequence and cation binding

“…However, it has recently been realised that binding may be more complex; individual antibodies recognising multiple HS structures. 16 This is consistent with the suggestion that a high degree of conformation- † 23 were both raised originally against bovine kidney HS and were selected for several reasons. They both recognised HS but by distinct epitopes, and their CDR-3 sequences, considered to be the major determinants of specificity, were very different, comprising SRKTRKPFMRK (6 basic residues out of 11) and HAPLRNTRTNT (2 basic residues plus histidine out of 11), respectively.…”

“…To examine the effects of cations on the binding of phage display antibodies to HS, a modification of an ELISA assay previously reported 16 was employed, in which the polysaccharides were first exposed to various cations before application of the phage display antibodies. Briefly, the polysaccharides were converted to the appropriate cation form by use of excess cation exchange resin (Dowex W-50) that had been converted from the acidic form to the appropriate cation form using a solution of the chloride of the metal ion concerned.…”

“…They both recognised HS but by distinct epitopes, and their CDR-3 sequences, considered to be the major determinants of specificity, were very different, comprising SRKTRKPFMRK (6 basic residues out of 11) and HAPLRNTRTNT (2 basic residues plus histidine out of 11), respectively. 16 …”

A Peptide Found by Phage Display Discriminates a Specific Structure of a Trisaccharide in Heparin

“…Verrecchio et al (24) reported that heparin-binding peptides were designed based on consensus sequences, (AKKARA) n and (ARKKAAKA) n (n ϭ 1-6), and then higher M r peptides 50 for inhibition of heparin-peptide binding was observed with 9 M heparin/HS, and chondroitin sulfate did not inhibit it. In addition, Schuksz et al (26) reported that the small molecule antagonist of heparan sulfate, surfen, bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin Ͼ dermatan sulfate Ͼ heparan sulfate Ͼ chondroitin sulfate.…”

“…Because almost no glycosaminoglycans are immunoreactive and there are few antibodies against them, this advantage is extremely useful for generating probes that bind to glycosaminoglycans. Thus, phage display technology is increasingly important for studying the functions of glycosaminoglycans (47)(48)(49)(50)(51)(52). The generation of type-specific anti-HS antibodies using phage display technology was initially applied by van Kuppevelt et al (53) for investigating HS heterogeneity in the kidney.…”